The Janus Kinase (JAK) Signal Transducer and Activator of Transcription (STAT) pathway is involved in the regulation of Intestinal Stem Cell (ISC) activity to ensure a continuous renewal of the adult Drosophila midgut. Three ligands, Unpaired1, 2 and 3 are known to activate the JAK/STAT pathway in Drosophila. Using newly generated upd mutants and cell specific RNAi, we showed that Upd1 is required throughout the fly life to maintain basal turnover of the midgut epithelium by controlling ISC maintenance in an autocrine manner. A role of Upd2 and Upd3 in basal conditions is discernible only in old gut, where they contribute to increased ISC abnormal division. Finally, upon an acute stress such as oral bacterial infection, we showed that Upd3 is released from enterocytes and has an additive effect with Upd2 to promote rapid epithelial regeneration. Taken together, our results show that Upd ligands are required to maintain the midgut homeostasis under both normal and pathological states.